Lymphokines are the proteins by which the immune cells communicate with each other. Scientists produce them in sufficient quantities for therapeutic use against immunologic diseases. The present invention relates particularly to previously unknown lymphokine and receptor proteins which were isolated and identified based on specific expression of the T cell genes using a technique identified by the present inventor in a publication (Proc. Natl. Acad. Sci. USA, 84, 2896-2900, May 1987, Immunology).
The immune system of humans and other species requires that white blood cells be made in the bone marrow, which white blood cells include phagocytes, lymphocytes and B cells. As presently understood, the phagocytes include macrophage cells which scavenge unwanted materials such as virus protein from the system. The lymphocytes include helper T cells and killer T cells and B cells as well as other cells, including those categorized as suppressor T cells.
The B cells produce the antibodies. The killer T cells physically pierce the cell and the helper T cells facilitate the whole process. In any event, the immune process is facilitated by lymphokines. Interleukin 1, secreted from macrophages activate the helper T cells and raise the body temperature causing fever which enhances the activity of the immune cells. The activated helper T cells produce Interleukin 2 and Interleukin stimulates the helper and killer T cells to grow and divide. The helper T cells also produce another lymphokine, B cell growth factor (BCGF), which causes B cells to multiply. As the number of B cells increases, the helper T cells produce another lymphokine known as the B cell differentiating factor (BCDF), which instructs some of the B cells to stop replicating and start producing antibodies. T cells also produce a lymphokine, gamma interferon (IF), which has multiple effects like Interleukin 2. Interferon helps activate killer T cells, enabling them to attack the invading organisms. Like BCGF, interferon increases the ability of the B cells to produce antibodies. Interferon also affects the macrophages to keep them at the site of the infection and help the macrophages to digest the cells they have engulfed. Gathering momentum with each kind of lymphokine signal between the macrophages and the T cells, the lymphokines amplify the immune system response and the virus protein or other foreign matter on the infected cells is overwhelmed. There are many other lymphokines, maybe a hundred or more, which participate in the immune process. Many lymphokines are known and many are not.
Lymphokines are sometimes called intercellular peptide signals. Among scientists there is widespread use of cloned cell lines as lymphokine producers and the isolation of lymphokine mRNA has become a common technique.
The protocol reported in the aforesaid publication can be used by scientists to detect virtually all of the lymphokines because the method is designed to detect virtually all the mRNA expressed differentially and the mRNA sequences of the immune cells are expressed differentially as they relate to the T cells and the killer T cells even though the level of expression is low and the quantity of the secreted lymphokine protein is low. The present inventor believes that the analysis described in the above identified publication can reveal biologically important molecules such as lymphokines because there are many indications that biologically important or active molecules are coded by the most scarce messages. An example is a transforming growth factor (TGF) which is present as only one of a million clones.
There are many known lymphokine proteins and they include the interferons, interleukin-1,2,3,4,5,6,7, colony-stimulating factors, lymphotoxin, tumor necrosis factor and erythropoietin, as well as others.
Most T cell factors have been classically identified by recognizing biologic activities in assays, purifying the protein information. An alternative approach is to isolate putative T cell genes based upon specific expression and then demonstrate the function of the unknown molecule. Using the aforesaid modified differential screening procedure, the present inventor has recently cloned a series of T cell subset-specific cDNAs from cloned helper T (HTL) L2 and cloned cytolytic T lymphocyte (CTL) L3.
Apparent full length cDNAs corresponding to fourteen species of the 16 initial isolates were sequenced and were found to constitute five different species.
Three of the five were identical to previously reported cDNA sequences of proenkephalin, T cell replacing factor and HF gene (a serine esterase). The other two, represented as L2G25B and 4-1BB, were novel sequences of unknown function. The open reading frames of 4-1BB and L2G25B code for 245 and 92 amino acids, respectively. The predicted proteins of 4-1BB and L2G25B include 22 and 23 amino acid-long putative signal sequences, respectively. The protein backbones of mature proteins encoded by 4-1BB and L2G25B are composed of 234 amino acids with molecular weight of 25000 and 69 amino acids with molecular weight of 7880, respectively. 4-1BB contains two potential N-glycosylation sites while L2G25B has none. 4-1BB contains 23 cysteine residues in the putative mature protein.
The cDNA L2G25B encodes for the lymphokine, macrophage inflammatory protein-1xcex1 or MIP-1xcex1. MIP-1xcex1 has been described in a paper entitled, xe2x80x9cEnhancing and Suppressing Effects of Recombinant Murine Macrophage Inflammatory Proteins on Colony Formation In Vitro by Bone Marrow Myeloid Progenitor Cellsxe2x80x9d, Hal E. Broxmeyer, Barbara Sherry, Li Lu, Scott Cooper, Kwi-Ok Oh, Patricia Tekamp-Olson, Byoung S. Kwon, and Anthony Cerami, Blood, 76 , 111-1116, 1990 and is incorporated herein by reference. This was the first time the suppressing activity of MIP-1xcex1 was characterized. Natural MIP-1 was found and characterized as an inflammatory protein in 1988 (Wolpe, S. D., G. Davatelis, B. Sherry, B. Beutler, D. G. Hesse, H. T. Hguyen, L. L. Moldawer, C. F. Nathan, S. F. Lowry, and A. Cerami. 1988. Macrophages secrete a novel heparin-binding protein with inflammatory and neutrophil chemokinetic properties. J. Exp. Med., 167, 570, incorporated herein by reference). The sequence for MIP-1xcex1 was also published in June 1988 (Davatelis, G., P. Tekamp-Olson, S. D. Wolpe, K. Hermsen, C. Luedke Gallegos, D. Cort, J. Merryweather, and A. Cerami. 1988. Cloning and characterization of a cDNA for murine macrophage inflammatory protein (MIP), a novel monokine with inflammatory and chemokinetic properties. J. Exp. Med., 167, 1939, incorporated herein by reference). The myelopoietic enhancing effects on colony formation in vitro by murine and human bone marrow granulocyte/macrophage progenitor cells was published in 1989 (Broxmeyer, H. E., B. Sherry, L. Lu, S. Cooper, C. Carow, S. D. Wolpe, and A. Cerami. 1989. Myelopoietic enhancing effects of murine macrophage inflammatory proteins and human bone marrow granulocyte/macrophage progenitor cells. J. Exp. Med., 170, 1583, incorporated herein by reference). In 1990, Graham and Pragnell identified and characterized an inhibitor of hematopoietic stem cell proliferation which turned out to be MIP-1xcex1. (Graham, G. J., E. G. Wright, R. Hewick, S. D. Wolpe, N. M. Wilke, D. Donaldson, S. Lorimore, and I. B. Pragnell. 1990. Identification and characterization of an inhibitor of haematopoietic stem cell proliferation. Nature, 344, 442, incorporated herein by reference). Graham and Pragnell have also published work on in vivo experiments of the suppressing effects of MIP-1xcex1 (David J. Dunlop, Eric G. Wright, Sally Lorimore, Gerald J. Graham, Tessa Holyoake, David J. Derr, Stephen D. Wolpe, and Ian B. Pragnell. 1992. Demonstration of Stem Cell Inhibition and Myeloprotective Effects of SCI/rhMIP-1xcex1 In Vivo, Blood, 79, No. 9, pp. 2221-2225, incorporated herein by reference).
The present invention includes the discovery of the cDNA sequence encoding for the protein later referred to as MIP-1xcex1 and the recombinant protein. A new use of a known human protein PLD78 has been discovered, wherein the new use is as a human lymphokine. The cDNA and fragments and derivatives thereof can be used as probes to isolate DNA sequences encoding for proteins similar to the lymphokine MIP-1xcex1. A method of suppressing progenitor cell cycling in a human patient prior to chemotherapy treatment has been developed. An appropriate dose of MIP-1xcex1 is administered to the patient to suppress early myeloid progenitor cell cycling and thereby suppress stem cell colony formation. Once the patient""s normal stem cell colonies are reduced, the chemotherapy treatment is administered. This method is based upon the work disclosed herein and the use of MIP-1xcex1 to suppress progenitor cell cycling in vivo in mice.
The effects of recombinant (r) mu, MIP-1xcex2 and MIP-2 on the suppressive activity of MIP-1xcex1 were tested using colony formation by hu and mu bone marrow BFU-E, CFU-GEMM and CFU-GM progenitor cells. MIP-1xcex2, but not MIP-2, when added with MIP-1xcex1 to cells, blocked the suppressive effects of MIP-1xcex1 on both hu and mu BFU-E, CFU-GEMM and CFU-GM colony formation. Similar results were observed regardless of the early acting cytokines used: rhuGM-CSF plus rhuIL-3, and two recently described potent cytokines, a genetically engineered rhuGM-CSF/IL-3 fusion protein and MGF, a c-kit ligand. The more potent the stimuli, the greater the suppressive activity noted. Pulse treatment of hu bone marrow cells with MIP-1xcex1 at 4xc2x0 C. for 1 hr. was as effective in inhibiting colony formation as continuous exposure of cells to MIP-1xcex1, and the pulsing effect with MIP-1xcex1 could not be overcome by subsequent exposure of cells to MIP-1xcex2. Also, pulse exposure of cells to MIP-1xcex2 blocked the activity of subsequently added MIP-1xcex1. For specificity, the action of a non-related myelosuppressive factor H-ferritin, was compared. MIP-1xcex1 and H-ferritin were shown to act on similar target populations of early BFU-E, CFU-GEMM and CFU-GM. MIP-1xcex2 did not block the suppressive activity of H-ferritin. Also, hemin and an inactive rhuH-ferritin mutein counteracted the suppressive effects of the wildtype H-ferritin molecule, but did not block the suppressive effects of MIP-1xcex1. These results show that MIP-1xcex2""s ability to block the action of MIP-xcex1 is specific. In addition, the results suggest that MIP-1xcex1 and MIP-xcex2 can, through rapid action, modulate early myeloid progenitor cell proliferation.
Recombinant proteins have been produced for the cytokine, L2G25BP (Macrophage Inflammatory Protein-1xcex1, MIP-1xcex1). By employing the recombinant protein (rMIP-1xcex1), receptors for MIP-1xcex1 were identified on Con A-stimulated and unstimulated CTLL-R8, a T-cell line, and LPS-stimulated RAW 264.7, a macrophage-cell line. The 125I-rMIP-1xcex1 binds to the receptor in a specific and saturable manner. Scatchard analysis indicated a single class of high affinity receptor with a Kd of approximately 1.5xc3x9710xe2x88x929M and approximately 1200 binding sites per Con A stimulated CTLL-R8 cell, and a Kd of 0.9xc3x9710xe2x88x929M and approximately 380 binding sites per RAW 264.7 cell. 125I-rMIP-1xcex1 binding was inhibited by unlabeled rMIP-1xcex1 in a dose-dependent manner, but not by IL-1xcex1 or IL-2. rMIP-1xcex1 inhibited the proliferation of unstimulated CTLL-R8 cells. Rabbit anti rMIP-1xcex1 antibodies blocked the growth inhibitory effect of the rMIP-1xcex1 on CTLL-R8 cells.
Purified recombinant murine macrophage inflammatory protein-1 alpha (rmuMIP-1xcex1), a cytokine with myelopoietic activity in vitro, was assessed in vivo by injection into C3H/HeJ mice for effects on proliferation (percentage of cells in S-phase) and absolute numbers of granulocyte-macrophage (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM) progenitor cells in the femur and spleen, and on nucleated cellularity in the bone marrow, spleen and blood. RmuMIP-1xcex1 rapidly decreased cycling rates (at 2-10 ug/mouse i.v.) and absolute numbers (at 5-10 ug/mouse i.v.) of myeloid progenitor cells in the marrow and spleen. These effects were dose- and time-dependent and reversible. Suppressive effects were noted within 3 to 24 hours for cell cycling and absolute numbers of progenitor cells in the marrow and spleen, and by 48 hours for circulating neutrophils. A study comparing the effects of i.v. injection of rmuMIP-1xcex1 versus rmuMIP-1xcex2, a biochemically similar molecule but with no myelosuppressive effects in vitro, demonstrated myelosuppression in vivo by rmuMIP-1xcex1 but not by rmuMIP-1xcex2. The results show that rmuMIP-1xcex1 has myelosuppressive activity in vivo and may be a used as an adjunct to treatments involving cytotoxic drugs because of its reversible suppressive effects on normal progenitor cell cycling.
Macrophage inflammatory protein-1xcex1 (MIP-1xcex1) is a member of the intercrine family which consists of basic, heparin-binding, small molecular weight proteins. A T-cell line CTLL-R8 cell was shown to carry high affinity receptors for MIP-1xcex1 and the proliferation of the CTLL-R8 cells was inhibited by murine recombinant (mr) MIP-1xcex1. Previous studies were extended to murine resting splenic T lymphocytes to determine whether the inhibition of T-cell proliferation is a general property of MIP-1xcex1. The resting splenic T-cells carried approximately 680 high affinity binding sites for mrMIP1xcex1. More than 90% of the primary T-cells carried the MIP-1xcex1 receptors. When the T-cells are stimulated with immobilized antiCD3 mAb in the presence of accessory cells, the MIP-1xcex1 receptor expression was down-regulated. The lowest binding was obtained 2 hrs after the antiCD3 mAb stimulation. This down-regulation was associated with the internalization of its own ligand which were produced upon antiCD3 mAb stimulation, mrMIP-1xcex1 inhibited the antiCD3 mAb-medicated proliferation of the murine splenic T lymphocytes. The maximum inhibition was obtained when the mrMIP-1xcex1 was added 30 min prior to the antiCD3 mAb stimulation. Slight inhibition of the T cell proliferation was observed when the mrMIP-1xcex1 was added 2 hrs prior to or at the same time as antiCD3 mAb stimulation. These results indicate that T lymphocytes are regulated negatively by MIP-l a, which occurs when the T-cells are exposed to MIP-1xcex1 prior to the activation. The negative effect of MIP-1xcex1 appears to be mediated by the inhibition of IL-2 production, for both IL-2 contents in the T-cell supernatant and IL-2 mRNA levels, were reduced.
The cDNA clone, called 4-1BB, was originally believed to be a lymphokine based upon the experiments disclosed herein. New studies show the 4-1BB is an inducible receptor-like sequence found in both cytolytic and helper T-cells. Polyclonal antibodies against oligopeptides representing the hydrophilic region of 4-1BB were prepared and used to characterize the properties of the molecule. The 4-1BB mRNA was detected in PMA-treated spleen and heart with constitutive expression detected in the kidney. FACS analysis and production of truncated, thus secretory, 4-1BB protein indicated that it is a cell-surface protein. The molecular size of 4-1BB protein was 40 kD. The 4-1BB protein was expressed on mononuclear cells infiltrating islet cells in the pancreata of NOD mice. Expression was prominent in the early phase of insulitis and the level of expression diminished or disappeared in the later phase. These data indicate that the 4-1BB protein is associated with T-cell activation and may play a role in the early phase of inflammation or autoimmune diseases.
The potential 4-1BB protein (4-1BBP) contains features seen in known receptor proteins and a region of amino acids similar to those in the nerve growth factor receptor, Drosophila gene sina, and Dictyostelium gene DG17. Polyclonal antibodies against a hydrophilic region of 4-1BBP were obtained and their distribution in the brain was examined. The specific expression pattern of 4-1BBP in the brain was identified in the gray matter where neuronal cell bodies, dendrites, and fiber terminals reside but was almost entirely absent in the white matter where axonal fibers dwell. A peculiar rosette pattern was observed in a granular layer of cerebellum and scattered in the stria terminalis. The staining pattern strongly resembled the receptor/nerve terminals in the brain and the peripheral nervous system. This study shows that the 4-1BBP is a novel receptor which may be associated with brain functions and is another example of a cell surface molecule found in both the immune and nervous systems.
The primary object is to provide the teachings identifying the new lymphokine and new receptor, L2G25B (MIP-1xcex1) and 4-1BB as identified herein by their gene sequence.
Another object of the present invention is to provide teachings of how the new lymphokine and new receptor may be used to isolate and identify corresponding molecules in related species.
Still another object of the teachings of the present invention is to teach the identification of the new lymphokine and new receptor as reported herein.